Assay for PhosphoRab activation of LRRK2 Kinase
Suzanne R Pfeffer, Claire Y Chiang
Abstract
MST kinase phosphorylates Rab proteins at the same site as LRRK2 and has been used to phosphorylate Rab8A and Rab10 quantitatively. This protocol includes a method to produce phosphoRab8A protein and remove as much contaminating MST3 as possible, to enable use of the phosphoRab to test subsequent activation of LRRK2 kinase. See these references for details on MST3 phosphorylation of Rab GTPases:
Steps
A. Phosphorylate Rab8A and remove MST3 kinase
To cleave the HIS tag from MST3, bind GST-PreScission protease (50µg) to 100µL glutathione agarose slurry (pre-washed and pelleted) 2h 0m 0s at 4°Cin a total volume of 500µL. Wash the resin 3X with 1mL reaction buffer. Add His-MST3 kinase (0.5mg) and incubate on a rotator at 4°C.
Spin beads 3000rpm,4°C and collect supernatant, which contains free MST3 and uncleaved His-MST3. Bind supernatant to Nickel-NTA agarose (100µL of a 50% slurry, prewashed and pelleted) for 2h 0m 0s at 4°C to trap uncleaved His-MST3.
Spin beads 3000rpm,4°C and collect supernatant, which contains free MST3 without the HIS tag.
Phosphorylate His-Rab8A Q67L full length with free MST3 using a molar ratio of 1:6 (kinase:substrate) at 30°C``2h 0m 0s in reaction buffer.
Phosphorylated His-Rab8A is then separated from MST3 by gel filtration using a 24mL Superdex 75 10/300 column (Cytiva Life Sciences, #17517401). Collect the relevant Rab-containing fractions determined by SDS-PAGE.
Further purify His-Rab8A by binding to Nickel-NTA agarose (100µL of a 50% slurry, prewashed and pelleted) and elute with 500millimolar (mM) imidazole (3 X 100µL) after washing with 60 column volumes of reaction buffer.
B. To measure phosphoRab activation of LRRK2 kinase:
Incubate 88nanomolar (nM) LRRK2 G2019S with 3micromolar (µM) His-GFP-Rab10 Q68L 1-181 or His-SUMO-Rab10 wild type full length substrate ± 6micromolar (µM) phosphoRab8A Q67L in reaction buffer in a total volume of 100µL.
Incubate reaction in a 30°C water bath and collect 20µL time points at 0, 10, and 20 minutes. Stop reactions with addition of 5µL SDS-PAGE sample buffer. Add 200nanomolar (nM) MLi-2 for control conditions to ensure that pRab10 detected is due to LRRK2 activity.
Analyze samples by SDS-PAGE and immunoblot for phosphoRab10; image blot with Li-COR and quantify bands using ImageJ (see below for more details).
